Electronic cigarettes heat a fluid containing nicotine and a humectant, such as propylene glycol. The resulting puff of aerosol contains many fewer chemicals than a puff of tobacco smoke and may therefore provide a less harmful alternative for those addicted to nicotine. However, relatively little is known about electronic cigarette aerosols and both positive and negative health effects have been reported by electronic cigarette users. Our long-term objective is to understand how electronic cigarettes affect human health and to contribute to the scientific foundation that will be used to establish rational policies for regulaing the manufacture, advertisement, and sale of electronic cigarettes. Our prior data demonstrate that some flavorings used in electronic cigarette refill fluids are cytotoxic to human cells. The goal of the proposed research is to compare the cytoxicity and genotoxicity of electronic cigarette aerosols and mainstream smoke from conventional cigarette products, and then to identify the flavorings and chemicals in electronic cigarette products that cause cytoxicity/genotoxicity. An in vitro toxicological approach will be used that will enable screening of broad range of products and identification of products/chemicals that could be pursued in more depth in future proposals including animals and humans. In our first Specific Aim, a dose response in vitro screen will be used to identify electronic cigarette products that produce aerosols which are cytotoxic to human embryonic stem cells, lung fibroblasts, and lung epithelial cells. A hierarchy of potency will establish the relative cytotoxicity of the products that are screened. Genotoxicity will then be evaluated at doses that do not kill cells. Similar cyto/genotoxicity data will be obtained using mainstream smoke from two conventional cigarette products. Data from the conventional and electronic cigarettes will be compared to determine if harm is decreased in the electronic cigarettes. Comparisons will also be made to determine the relative sensitivity of embryonic vs. adult cells to these products. In the second Specific Aim, we will identify the chemicals in the most potent electronic cigarette aerosols, purchase authentic standards, and test the authentic standards in the cytotoxic and genotoxic assays to identify the chemical(s) that produce harm. The resulting data would contribute to regulatory science and help regulatory agencies establish policies to govern the chemicals and flavorings that are added to electronic cigarette fluids. These data could help prevent adverse health effects in electronic cigarette users and make electronic cigarettes a safer alternative to conventional tobacco products.